Superoxide release and cellular
glutathione peroxidase activity
in leukocytes from children with
persistent asthma
1Departamento de Pediatria e Centro de Investigação em Pediatria,
Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, SP, Brasil
2Department of Pediatrics, University of Massachusetts Medical School,
Worcester, MA, USA L.E. Marçal1, J. Rehder1,
P.E. Newburger2 and
A. Condino-Neto1
Abstract
Asthma is an inflammatory condition characterized by the involve-ment of several mediators, including reactive oxygen species. The aim of the present study was to investigate the superoxide release and cellular glutathione peroxidase (cGPx) activity in peripheral blood granulocytes and monocytes from children and adolescents with atopic asthma. Forty-four patients were selected and classified as having intermittent or persistent asthma (mild, moderate or severe). The spontaneous or phorbol myristate acetate (PMA, 30 nM)-induced superoxide release by granulocytes and monocytes was determined at 0, 5, 15, and 25 min. cGPx activity was assayed spectrophotometri-cally. The spontaneous superoxide release by granulocytes from pa-tients with mild (N = 15), moderate (N = 12) or severe (N = 6) asthma was higher at 25 min compared to healthy individuals (N = 28, P < 0.05, Duncan test). The PMA-induced superoxide release by granulo-cytes from patients with moderate (N = 12) or severe (N = 6) asthma was higher at 15 and 25 min compared to healthy individuals (N = 28, P < 0.05 in both times of incubation, Duncan test). The spontaneous or PMA-induced superoxide release by monocytes from asthmatic pa-tients was similar to healthy individuals (P > 0.05 in all times of incubation, Duncan test). cGPx activity of granulocytes and mono-cytes from patients with persistent asthma (N = 20) was also similar to healthy individuals (N = 10, P > 0.05, Kruskal-Wallis test). We conclude that, under specific circumstances, granulocytes from chil-dren with persistent asthma present a higher respiratory burst activity compared to healthy individuals. These findings indicate a risk of oxidative stress, phagocyte auto-oxidation, and the subsequent release of intracellular toxic oxidants and enzymes, leading to additional inflammation and lung damage in asthmatic children.
Correspondence
A. Condino-Neto
Departamento de Pediatria e Centro de Investigação em Pediatria FCM, UNICAMP Caixa Postal 6111 13081-970 Campinas, SP Brasil
Fax: +55-19-3289-8638 E-mail: [email protected] or [email protected]
Research supported by FAPESP (No. 98/12144-5) and Fogarty International Center, United States National Institutes of Health (RO3-TW00883). L.E. Marçal was supported by a FAPESP fellowship (No. 97/13954-9).
Received October 31, 2003 Accepted August 17, 2004
Key words
Introduction
The major characteristics of asthma are reversible airflow obstruction, bronchial hy-perresponsiveness, and airway inflammation (1). This is a complex inflammatory disease that involves leukocytes, airway epithelial and smooth muscle cells, and several inflam-matory mediators with multiple effects. The airway remains edematous and infiltrated with inflammatory cells, which are predomi-nantly eosinophils and lymphocytes. Mast cells play a key role in asthma symptoms, whereas eosinophils, macrophages and T-helper 2 cells are involved in the chronic inflammation that underlies airway hyperre-sponsiveness (2). Resident or infiltrating in-flammatory phagocytes in the airways re-lease reactive oxygen species and other me-diators with pleiotropic effects (1).
The role of phagocytes in the pathophysi-ology of asthma is not completely under-stood. They release enzymes, including ex-tracellular matrix-degrading proteases, elas-tase, cytokines, and reactive oxygen species, which have been implicated in lung injury (3). The reported effects of reactive oxygen species in asthma include a decrease of beta-adrenergic function in lungs, airway smooth muscle contraction, increased vascular per-meability, bronchial hyperresponsiveness, increased mucus secretion, impaired ciliary activity, generation of chemotactic factors, lipid peroxidation, and secondary produc-tion of mediators with a bronchoconstrictor effect (4,5). Airway inflammation is also associated with increased activity of the in-ducible nitric oxide synthase found in respi-ratory epithelium and activated macrophages (6). Superoxide and nitric oxide may rapidly combine to form peroxynitrite, a potent oxi-dant, leading to the depletion of antioxi-dants. For instance, the antioxidant glutathi-one (GSH), which is 100-fold more concen-trated in the airway epithelial lining fluid compared with plasma, may be converted to its oxidized form (GSSG) (7). Thus,
subse-quent lung injury and more inflammation may take place.
Glutathione peroxidases (GPx) play an important role in the detoxificationof vari-ous hydroperoxides. Four types of GPx have been identified: cellularGPx (cGPx), gas-trointestinal GPx, extracellular GPx,and phospholipid hydroperoxide GPx (8,9). cGPx (EC 1.11.1.9), also termed GPX1, is ubiqui-tously distributed. It reduces hydrogen per-oxide as well as a wide range of organic peroxides derived from unsaturated fatty acids, nucleic acids, and other important biomolecules (8). At peroxide concentra-tions encountered under physiological con-ditions, it is more active than catalase (which has a higher Km for hydrogen peroxide) and
is also active against organic peroxides. Thus, cGPx represents a major cellular defense against toxic oxidant species (9).
The ability of cGPx to reduce peroxides and hydroperoxides plays a particularly im-portant role in the anti-oxidant defense sys-tem of phagocytes, which are subject to auto-oxidation by their own respiratory burst prod-ucts (10,11), leading to damage of the sur-rounding tissues when a great amount of oxygen radicals are released (12). Thus, oxi-dative stress is a relevant risk factor for lung damage in chronic inflammatory diseases such as asthma (6,13).
The aim of the present study was to in-vestigate superoxide release and cGPx activ-ity of granulocytes and monocytes from chil-dren and adolescents with atopic asthma, classified according to the Global Initiative for Asthma (GINA) criteria for evaluation of disease severity.
Subjects and Methods
Subjects and study design
(mean = 10.8 ± 2.85 years, median 11 years). The height ranged from 115 to 173 cm, and weight from 21 to 59 kg. Our clinical proto-col included patients with a combination of clinical history of recurrent and reversible symptoms of airway obstruction, high serum IgE levels, positive skin tests to recognized allergens, and a family history of allergy (14). We were not allowed to collect blood samples from healthy children. Thus, we selected 28 healthy individuals for the com-parative group, with an age range of 22-43 years.
The experimental protocol included as-says of superoxide production and cGPx activity in peripheral blood granulocytes and monocytes. Written informed consent was obtained from all patient’s parents and healthy individuals prior to the study. The Medical School Ethics Committee approved the experimental protocol in accordance to the Ministry of Health of Brazil (resolution 196/96) and the Helsinki Convention.
Severity of disease was classified ac-cording to GINA criteria into mild intermit-tent, mild persisintermit-tent, moderate persisintermit-tent, and severe persistent (15). Patients with moderate persistent asthma used inhaled beta2-adrenergic and/or inhaled steroids
(equivalent to 400-800 µg/day budesonide). Patients with severe persistent asthma used inhaled beta2-adrenergic and inhaled, but
not systemic steroids (equivalent to 800-1600 µg/day budesonide).
None of the patients had received non-steroidal anti-inflammatory drugs, theophyl-line, leukotriene modifiers, systemic ster-oids, systemic beta2-adrenergic agents, or
blood transfusion for at least one month prior to the study. Patients with infections, airway foreign bodies, or known systemic or pulmonary chronic diseases such as immu-nodeficiency, diabetes, ciliary dyskinesia syndromes, cystic fibrosis, or alpha1
-anti-trypsin deficiency were excluded. Smokers were excluded from both patient and control groups.
Isolation of granulocytes and monocytes
Granulocytes and monocytes were iso-lated by centrifugation of blood samples over a discontinuous density gradient (Histopaque 1.077 and 1.119 g/ml; Sigma, St. Louis, MO, USA) followed by adherence of monocytes to polystyrene plates (16). Contaminating erythrocytes were removed by hypotonic ly-sis. Both cell populations were washed three times in Hank’s balanced salt solution (HBSS) without phenol red and the final leukocyte count was adjusted to 2 x 107
cells/ml. Trypan blue exclusion showed greater than 90% cell viability.
Superoxide anion production
The spontaneous or phorbol myristate acetate (PMA, 30 nM)-induced superoxide anion production by leukocytes was assayed by a spectrophotometric method based on the superoxide dismutase inhibitable reduc-tion of cytochrome c, according to McCord and Fridovich (17), as modified (18). The absorbance at 550 nm of the supernatants was monitored at 0, 5, 15, and 25 min. The amount of superoxide was calculated using an extinction coefficient of 21,100 M/cm. The results are reported as nmol of superox-ide released per 106 cells per sampling time.
This assay was performed in granulocytes and monocytes from 44 patients and 28 healthy individuals.
Cellular glutathione peroxidase activity
cGPx activity was assayed in peripheral blood granulocytes and monocytes accord-ing to the method of Beutler (19), as modi-fied (20). Leukocytes (106) were incubated
Results are reported as nmol of oxidized NADPH min-1 106 cells-1. This assay was
performed in granulocytes and monocytes from 20 patients and 10 healthy individuals according to cell number availability.
Statistical analysis
Comparisons between the patient groups and healthy individuals were made using the Duncan or Kruskal-Wallis (21) test as
indi-cated, with the level of significance set at P < 0.05.
Results
The spontaneous superoxide production by granulocytes from severe, moderate or mild persistent asthmatic patients was higher at 25 min of incubation compared to healthy individuals (Figure 1A, P < 0.05, Duncan test).
nmol O
2
-/10
6 granulocytes
5.0 4.5 2.0 1.5 0.5 0 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 4.0 3.5 3.0 2.5 1.0
0 5 15 25
Time (min) 16 14 6 4 0 12 10 8 2
0 5 15 25
Time (min)
Healthy 12 12
MI MIP MOP SP
123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 * * * * A B Figure 1. Superoxide release by
granulocytes and monocytes from patients with asthma, grouped according to GINA di-agnostic criteria. Panel A, Spon-taneous superoxide (O2-) pro-duction was measured in granu-locytes from patients with asthma grouped according to GINA criteria and from healthy individuals at the indicated time points. The spontaneous super-oxide production by granulo-cytes from SP, MOP or MIP asthmatic patients was higher at 25 min of incubation, com-pared to healthy individuals (*P < 0.05, Duncan test). Panel B, Phorbol myristate acetate (PMA)-stimulated O2- production was measured in granulocytes from patients with asthma grouped according to GINA cri-teria and from healthy individu-als at the indicated time points. The PMA-stimulated superoxide production by granulocytes from SP and MOP asthmatic patients was higher compared to healthy individuals at 15 and 25 min of incubation (*P < 0.05, Duncan test). SP = severe persistent (N = 6), MOP = moderate ent (N = 12), MIP = mild persist-ent (N = 15), and MI = mild inter-mittent asthma (N = 11). Healthy
individuals (N = 28). nmol O
2
-/10
6 granulocytes
The PMA-stimulated superoxide produc-tion by granulocytes from severe and moder-ate persistent asthmatic patients was higher compared to healthy individuals at 15 and 25 min of incubation (Figure 1B, P < 0.05, Duncan test).
However, neither spontaneous nor PMA-stimulated superoxide production by mono-cytes from asthmatic patients, grouped ac-cording to GINA criteria, differed statisti-cally from healthy individuals (P > 0.05, Duncan test).
The cGPx activity of granulocytes from mild persistent (N = 12), moderate persistent and severe persistent asthmatic children (N = 8), and healthy individuals (N = 10) were (mean ± SD, nmol of oxidized NADPH min-1
106 cells-1): 171 ± 75, 181 ± 33, and 197 ± 68,
respectively. The cGPx activity of mono-cytes from mild persistent (N = 12), moder-ate persistent and severe persistent asthmatic children (N = 8), and healthy individuals (N = 10) were (mean ± standard deviation, nmol of oxidized NADPH min-1 106 cells-1): 142 ±
52, 151 ± 75, and 136 ± 36, respectively. Thus, the cGPx activity of granulocytes or monocytes from children and adolescents with persistent asthma did not differ from that of healthy individuals (P > 0.05 in all situations, Kruskal-Wallis test).
Discussion
Our results show that the spontaneous or stimulated superoxide production by granu-locytes from children and adolescents with persistent asthma is elevated compared to healthy individuals.
Ethical restrictions were much greater than ordinary because the study concerned samples from children. We were not allowed to obtain bronchoalveolar lavage cells from our patients with asthma, an apparently more sensitive approach to the questions we asked. Thus, we chose to investigate the superoxide release and cGPx activity of blood leuko-cytes. Indeed, one must consider that both
bronchoalveolar lavage cells and blood leu-kocytes are not intralesional lung tissue cells. In addition, we were also not allowed to collect blood samples from healthy children. Thus, we selected 28 healthy individuals for the comparative group, which could not be age matched. Previous studies have shown that the respiratory burst activity of leuko-cytes from children and adolescents does not differ significantly from that of adults (22,23). This similarity allowed us to compare the NADPH oxidase activity of leukocytes from children with asthma to measurements in healthy adult individuals.
Considering the limitation of volume when collecting blood samples from chil-dren, we were unable to separate neutrophils (predominant cells), eosinophils and baso-phils from the granulocyte pool. Thus, we chose to perform those tests in granulocytes. It is generally accepted that eosinophils and neutrophils produce equivalent amounts of superoxide, although this is still a controver-sial issue. Because of differences in the as-sembly of the NADPH oxidase components, eosinophils show a trend to release more extracellular superoxide compared to neu-trophils (24).
Other studies have demonstrated that leu-kocytes from asthmatic patients show higher superoxide production compared to healthy individuals, particularly during asthma at-tacks (6,24-31). In the present, more de-tailed, study, pediatric patients with chronic asthma were for the first time carefully clas-sified according to GINA criteria. In addi-tion, we have assessed both respiratory burst and cGPx activity in granulocytes and mono-cytes. Only patients with persistent asthma showed a significant increase in superoxide release by granulocytes. These findings dem-onstrate at least a partial relationship be-tween asthma severity and respiratory burst activity in peripheral blood granulocytes from pediatric patients.
ac-cording to cell lineage or body fluid. GPx activity may be lower in platelets, normal or lower in red blood cells, lower or increased in plasma, and normal or lower in whole blood (6,13,26,32,33). These conflicting data may be the result of assaying distinct iso-forms of GPx in different blood components from heterogeneous asthmatic patients. To date, we have shown that cGPx activity of leukocytes from pediatric asthmatic patients, carefully classified according to GINA crite-ria, was similar to healthy controls and did not follow the up-regulation of NADPH oxi-dase activity in the same cells.
We conclude that under specific circum-stances granulocytes from children and ado-lescents with persistent asthma produce more reactive oxygen species compared to healthy individuals. These reactive species have the
potential to overwhelm the antioxidant sys-tem, with resultant oxidative stress and dam-age of surrounding pulmonary tissue. This process may be a relevant and still uncon-trolled risk factor in childhood asthma patho-physiology. In children with asthma, key markers of inflammation are present early in life, highlighting the importance of early intervention and the appropriate use of drugs that modulate the NADPH oxidase activity (34), preventing the irreversible lung remod-eling that contributes to this chronic disease.
Acknowledgments
The authors thank Drs. Marluce Vilela, José Dirceu Ribeiro, Adyléia Toro, and Cristina Sartorelli for helpful discussions and patient care.
References
1. Busse WW & Lemanske Jr RF (2001). Advances in immunology: asthma. New England Journal of Medicine, 344: 350-362. 2. Barnes PJ (2003). Pathophysiology of asthma. European
Respira-toryMonograph, 23: 84-113.
3. Vignola AM, Bonanno A, Mirabella A, Riccobono L, Mirabella F, Profita M, Bellia V, Bousquet J & Bonsignore G (1998). Increased levels of elastase and alpha1-antitrypsin in sputum of asthmatic patients. American Journal of Respiratory and Critical Care Medi-cine, 157: 505-511.
4. Hancock JT (1997). Superoxide, hydrogen peroxide and nitric oxide as signaling molecules; their production and role in disease. British Journal of Biomedical Science, 54: 38-46.
5. Abraham WM (1997). Reactive oxygen species. In: Barnes PJ, Grunstein MM, Leff AR & Woolcock AJ (Editors), Asthma. Lippin-cott-Raven Publishers, Philadelphia, PA, USA, 627-638.
6. Bowler RP & Crapo JD (2002). Oxidative stress in airways. Is there a role for extracellular superoxide dismutase? American Journal of Respiratory and Critical Care Medicine, 166: 38-43.
7. Van der Vliet A, O’Neill CA, Cross CE, Koostra JM, Volz WG, Halli-well B & Louie S (1999). Determination of low-molecular-mass antioxidant concentrations in human respiratory tract lining fluids.
American Journal of Physiology, 276: L289-L296.
8. Tappel AL (1984). Selenium-glutathione peroxidase: properties and synthesis. Current Topics in Cellular Regulation, 24: 87-97. 9. Takebe G, Yarimizu J, Saito Y, Hayashi T, Nakamura H, Yodoi J,
Nagasawa S & Takahasi K (2002). A comparative study on the hydroperoxide and thiol specificity of the glutathione peroxidase family and selenoprotein P. Journal of Biological Chemistry, 277: 41254-41258.
10. Baehner RL, Boxer LA, Allen JM & Davis J (1977). Autooxidation as a basis for altered function by polymorphonuclear leukocytes. Blood, 50: 327-335.
11. Ito Y, Kajkenova O, Feuers RJ, Udupa KB, Desai VG, Epstein J, Hart RW & Lipschitz DA (1998). Impaired glutathione peroxidase activity accounts for the age-related accumulation of hydrogen peroxide in activated human neutrophils. Journal of Gerontology. Series A, Biological Sciences and Medical Sciences, 53: M169-M175. 12. Boxer LA (1990). The role of antioxidants in modulating neutrophil
functional responses. Advances in Experimental Medicine and Biol-ogy, 262: 19-33.
13. Rahman I & MacNee W (2000). Oxidative stress and regulation of glutathione in lung inflammation. European Respiratory Journal, 16: 534-554.
14. Condino-Neto A, Vilela MM, Cambiucci EC, Ribeiro JD, Guglielmi AAG, Magna LA & De Nucci G (1991). Theophylline therapy inhibits neutrophil and mononuclear cell chemotaxis from chronic asth-matic children. British Journal of Clinical Pharmacology, 32: 557-561.
15. National Heart, Lung and Blood Institute(1997). Practical Guide for the Diagnosis and Management of Asthma. Expert Panel Report 2, Publication 97-4053.
16. Boyum A (1968). Isolation of mononuclear cells and granulocytes from human blood. Scandinavian Journal of Clinical and Laboratory Investigation, 21 (Suppl 97): 77-89.
17. McCord J & Fridovich I (1969). Superoxide dismutase: An enzymatic function for erythrocuprein (hemocuprein). Journal of Biological Chemistry, 244: 6049-6055.
and cytochrome b558 content of human Epstein-Barr-virus-trans-formed B lymphocytes correlate with expression of genes encoding components of the oxidase system. Archives of Biochemistry and Biophysics, 360: 158-164.
19. Beutler E (1984). Glutathione peroxidase. In: Beutler E (Editor), Red Cell Metabolism. Grune & Stratton, Orlando, CA, USA, 74-76. 20. Speier C, Baker SS & Newburger PE (1985). Relationships between
in vitro selenium supply, glutathione peroxidase activity, and phago-cytic function in the HL-60 human myeloid cell line. Journal of Biological Chemistry, 260: 8951-8955.
21. Montgomery DC (1991). Design and Analysis of Experiments. 3rd edn. John Wiley & Sons, New York.
22. Drossou V, Kanakoudi F, Tzimouli V, Sarafidis K, Taparkou A, Bougiouklis D, Petropulou T & Kremenipoulos G (1997). Impact of prematurity, stress and sepsis on the neutrophil respiratory burst activity of neonates. Biology of the Neonate, 72: 201-209. 23. Speer CP, Ambruso DR, Grimsley J & Johnston Jr RB (1985).
Oxidative metabolism in cord blood monocytes and monocyte-derived macrophages. Infection and Immunity, 50: 919-921. 24. Lacy P, Abdel-Latif D, Steward M, Musat-Marcu S, Man SF &
Moqbel R (2003). Divergence of mechanisms regulating respiratory burst in blood and sputum eosinophils and neutrophils from atopic subjects. Journal of Immunology, 170: 2670-2679.
25. Jarjour NN & Calhoun WJ (1994). Enhanced production of oxygen radicals in asthma. Journal of Laboratory and Clinical Medicine, 123: 131-136.
26. Bowler RP & Crapo JD (2002). Oxidative stress in allergic respira-tory diseases. Journal of Allergy and Clinical Immunology, 110: 349-356.
27. Kanazawa H, Kurihara N, Hirata K & Takeda T (1991). The role of free radicals in airway obstruction in asthmatic patients. Chest, 100: 1319-1322.
28. Cluzel M, Damon M, Chanez P, Bousquet J, Crastes de Paulet A, Michel FB & Godard P (1987). Enhanced alveolar cell luminol-de-pendent chemiluminescence in asthma. Journal of Allergy and Clini-cal Immunology, 80: 195-201.
29. Vachier I, Damon M, Le Doucen C, Crastes de Paulet A, Chanez P, Michel FB & Godard P (1992). Increased oxygen species generation in blood monocytes of asthmatic patients. American Review of Respiratory Diseases, 146: 1161-1166.
30. Sedgwick JB, Geiger KM & Busse WW (1990). Superoxide genera-tion by hypodense eosinophils from patients with asthma. Ameri-can Review of Respiratory Diseases, 142: 120-125.
31. Monteseirin J, Camacho MJ, Bonilla I, De la Calle A, Guardia P, Conde J & Sobrino F (2002). Respiratory burst in neutrophils from asthmatic patients. Journal of Asthma, 39: 619-624.
32. Shanmugasundaram KR, Kumar SS & Raajjee S (2001). Excessive free radical generation in the blood of children suffering from asthma. Clinica Chimica Acta, 305: 107-114.
33. Nadeem A, Chhabra SK, Masood A & Raj HG (2003). Increased oxidative stress and altered levels of antioxidants in asthma. Jour-nal of Allergy and Clinical Immunology, 111: 72-78.
